IL-12 an adjuvant for bordetella pertussis vaccines

ABSTRACT

This invention provides a composition of at least one  Bordetella  antigen and an effective adjuvant amount of interleukin-12 (IL-12), and uses thereof as a vaccine against  Bordetella  infection. Methods for using IL-12 as an adjuvant in combination with vaccines against  Bordetella  are also provided.

This application claims priority from provisional application60/015,871, filed May 31, 1996.

BACKGROUND OF THE INVENTION

The present invention relates generally to vaccines against Bordetellaspecies that include interleukin-12 (IL-12) as an adjuvant, and tomethods for using IL-12 as an adjuvant in or in combination with suchvaccines.

Colonization of the respiratory tract by the Gram-negative coccobacillusBordetella pertussis results in whooping cough, also called pertussis, asignificant cause of morbidity and mortality of human infants. Two otherclosely-related isolates of Bordetella have also been found in humans:B. parapertussis and B. bronchiseptica. Molecular genetic analysessuggest that these three isolates are too closely related to beclassified as separate species. (Gilchrist. M. J. R., 1991,“Bordetella”, in Manual of Clinical Microbiology, 5th ed., Balows, A. etal., eds., American Society for Microbiology, Washington, D.C.) While B.pertussis differs from B. bronchiseptica and B. parapertussis in thenature of the toxins it produces, B. bronchiseptica and B. parapertussisdo produce active toxins (Hausman, S. Z. et al., 1996, Infect. Immun.64: 4020-4026), and there is some evidence to indicate that B. pertussisorganisms can covert to the B. parapertussis phenotype (Gilchrist, M. J.R., 1991, “Bordetella”, in Manual of Clinical Microbiology, 5th ed.,Balows, A. et al., eds., American Society for Microbiology, Washington,D.C.). Although Bordetella isolates exhibit some surface antigens thatdiffer between isolates, monoclonal antibodies that recognize oneisolate often recognize at least one other isolate (LeBlay, K. et al.,1996, Microbiology 142: 971-978). The high degree of molecularsimilarity between Bordetella isolates and the cross-reactivity ofmonoclonal antibodies to Bordetella antigens indicates that the immuneresponse produced by a vaccine against one Bordetella isolate wouldlikely affect the other isolates as well.

Immunization with a whole-cell Bordetella pertussis vaccine has provedefficacious in controlling pertussis, but concern has been raised overits reactogenicity. Pertussis acellular vaccines are significantly lessreactogenic but are of varying efficacy. Until recently the bacteriumwas thought to occupy a purely extracellular niche during infection andconsequently humoral immune mechanisms were assumed to be paramount inprotection. (Robinson, A. et al., 1985, Vaccine 3: 11-22.) However,there is increasing evidence from human and murine studies that B.pertussis can also occupy an intracellular niche through invasion andsurvival within lung macrophages and other cell types. (Friedman, R. L.et al., 1992, Infect. Immun. 60: 4578-4585; Saukkonen, K. et al., 1991,J. Exp. Med. 173: 1143-1149.) These observations have led to areexamination of the mechanisms of protective immunity against B.pertussis. While antibody plays a role in bacterial toxin neutralizationand in the prevention of bacterial attachment following transudation ofcirculating immunoglobulin (Ig) into the lung, cell-mediated immunityalso plays a significant role in protection against B. pertussis.(Mills, K. H. G. and K. Redhead, 1993, J. Med. Microbiol. 39: 163-164;Peppoloni, S. et al., 1991, Infect. Immun. 59: 3768-3773; Peterson, J.P. et al., 1992, Infect. Immun. 60: 4563-4570.)

The current understanding of the role of CD4⁺ T helper (Th) cells inimmunity to infectious diseases is that antigen-specific type 1 T helper(Th1) cells which secrete interferon-γ (IFN-γ), interleukin-2 (IL-2),and tumor necrosis factor-β (TNF-β) mediate cellular immunity,delayed-type hypersensitivity, and inflammatory responses, whereas type2 T helper (Th2) cells which secrete the interleukins IL-4, IL-5, andIL-6 are considered to be mainly responsible for the provision ofspecific T cell help for antibody production. (Mosmann, T. R. and R. L.Coffman, 1989, Adv. Immunol. 46: 111-147.) Previous studies using amurine respiratory model have demonstrated that protective immunityagainst B. pertussis induced by infection is mediated by a CD4⁺ T cellpopulation that secreted IL-2 and IFN-γ (Th1 cells). Adoptive transferexperiments demonstrated that protection could be conferred with T cellsin the absence of detectable antibody responses. In a study ofvaccine-induced immunity, immunization with the whole-cell pertussisvaccine selectively induced Th1 cells, whereas an acellular vaccine,comprising the B. pertussis antigens detoxified PT, FHA, and pertactin,induced Th2 cells. Furthermore, the induction of a Th1 responsefollowing infection or immunization with the whole-cell vaccine wasassociated with earlier bacterial clearance following respiratorychallenge. (Mills, K. H. G. et al., 1993, Infect. Immun. 61: 399-410;Redhead, K. et al., 1993, Infect. Immun. 61: 3190-3198.)

The polarization of CD4⁺ T cell cytokine production towards type 1 ortype 2 responses following in vivo priming appears to be controlled by anumber of factors including the nature of the immunogen, the route ofimmunization, and the antigen-presenting cell and regulatory cytokinemilieu at the site of T cell stimulation. (Barnard, A. et al., 1996,Immunol. 87: 372-380; Gajewski, T. F. et al., 1991, J. Immunol. 146:1750-1758; O'Gara, A. and K. Murphy, 1994, Curr. Opin. Immunol. 6:458-466.) The regulatory cytokine interleukin-12 (IL-12) is also a keycytokine in the development of type 1 responses. (Hsieh, C.-S. et al.,1993, Science 260: 547-549; Trinchieri, G., 1995, Annu. Rev. Immunol.13: 251-276.) IL-12 can induce the secretion of IFN-γ by natural killer(NK) cells and by CD4⁺ T cells and can promote the differentiation anddevelopment of Th1 cells from Th0 precursor populations. (Bliss, J. etal., 1996, J. Immunol. 156: 887-894; McKnight, A. J. et al., 1994, J.Immunol. 152: 2172-2179; Seder, R. A. et al., 1993, PNAS USA 90:10188-10192.) Furthermore, IL-12 may also induce the production ofopsonizing antibodies, by promoting IFN-γ-mediated immunoglobulin (Ig)class switching in favor of IgG2a in the mouse. (Morris, S. C. et al.,1994, J. Immunol. 152: 1047-1056.) Since Th1 cells play an importantrole in the resolution of infections with intracellular organisms, IL-12can influence the course of bacterial, viral, and parasitic infectionsby altering the balance of Th1 and Th2 cells in favor of IFN-γproduction. (Flynn, J. L. et al., 1995, J. Immunol. 155: 2515-2524;Gazzinelli, R. T. et al., 1993, PNAS USA 90: 6115-6119; Heinzel, F. P.et al., 1993, J. Exp. Med. 177: 1505-1509; Hunter, C. A. et al., 1994,Infect. Immun. 62: 2818-2824; Sypek, J. P. et al., 1993, J. Exp. Med.177: 1797-1802; Tripp, C. S. et al., 1994, J. Immunol. 152: 1833-1887;Urban, J. F. et al., 1996, J. Immunol. 156: 263-268; Wynn, T. A. et al.,1994, J. Exp. Med. 179: 1551-1561; Zhan, Y. and C. Cheers, 1995, Infect.Immun. 63: 1387-1390.)

There is a continuing requirement for new compositions comprising IL-12that will enhance or alter the effects of Bordetella vaccines, and formethods for their use in the prevention, treatment, or amelioration ofBordetella infections.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the use of IL-12 asan adjuvant in an acellular Bordetella vaccine significantly increasedits protective efficacy.

In one embodiment, the present invention provides a compositioncomprising at least one Bordetella antigen and an effective adjuvantamount of interleukin-12. Preferably, the antigen is a Bordetellapertussis antigen, or is lipopolysaccharide, pertussis toxin,filamentous hemagglutinin, or pertactin, or is adsorbed to alum.

In another embodiment, the invention provides a composition comprisingan effective adjuvant amount of interleukin-12 and at least oneantigen-encoding polynucleotide capable of expression in vivo to produceat least one Bordetella antigen.

A further embodiment provides a composition comprising at least oneBordetella antigen and an interleukin-12-encoding polynucleotide capableof expression in vivo to produce an effective adjuvant amount ofinterleukin-12.

Another embodiment provides a method for preventing, treating, orameliorating infection by Bordetella in a host, comprising administeringto the host a composition comprising at least one Bordetella antigen andan effective adjuvant amount of interleukin-12. Preferably, the antigenis a Bordetella pertussis antigen, or is lipopolysaccharide, pertussistoxin, filamentous hemagglutinin, or pertactin, or is adsorbed to alum,or is administered as an antigen-encoding polynucleotide underconditions in which the antigen is expressed in vivo. Preferably, theinterleukin-12 may be administered as an interleukin-12-encodingpolynucleotide under conditions in which the interleukin-12 is expressedin vivo.

In another embodiment, the invention provides a method for eliciting animmune response against Bordetella comprising administering acomposition comprising at least one Bordetella antigen and an effectiveadjuvant amount of interleukin-12. Preferably, the antigen is aBordetella pertussis antigen, or is lipopolysaccharide, pertussis toxin,filamentous hemagglutinin, or pertactin, or is adsorbed to alum, or isadministered as an antigen-encoding polynucleotide under conditions inwhich the antigen is expressed in vivo. Preferably, the interleukin-12may be administered as an interleukin-12-encoding polynucleotide underconditions in which the interleukin-12 is expressed in vivo.

In a further embodiment, the present invention provides a method foreliciting an immune response against Bordetella comprising administeringsimultaneously a first composition comprising at least one Bordetellaantigen and a second composition comprising an effective adjuvant amountof interleukin-12. Preferably, the antigen is a Bordetella pertussisantigen, or is lipopolysaccharide, pertussis toxin, filamentoushemagglutinin, or pertactin, or is adsorbed to alum, or is administeredas an antigen-encoding polynucleotide under conditions in which theantigen is expressed in vivo. Preferably, the interleukin-12 may beadministered as an interleukin-12-encoding polynucleotide underconditions in which the interleukin-12 is expressed in vivo.

Another embodiment of the present invention provides a method forstimulating clearance of Bordetella from a host comprising administeringa composition comprising at least one Bordetella antigen and aneffective adjuvant amount of interleukin-12. Preferably, the antigen isa Bordetella pertussis antigen, or is lipopolysaccharide, pertussistoxin, filamentous hemagglutinin, or pertactin, or is adsorbed to alum,or is administered as an antigen-encoding polynucleotide underconditions in which the antigen is expressed in vivo. Preferably, theinterleukin-12 may be administered as an interleukin-12-encodingpolynucleotide under conditions in which the interleukin-12 is expressedin vivo.

A further embodiment provides a method for preparing an improved vaccinecomposition comprising combining an effective adjuvant amount ofinterleukin-12 with a vaccine composition comprising at least oneBordetella antigen. Preferably, the antigen is a Bordetella pertussisantigen, or is lipopolysaccharide, pertussis toxin, filamentoushemagglutinin, or pertactin, or is adsorbed to alum.

A method is also provided for preparing an improved vaccine compositioncomprising combining an effective adjuvant amount of interleukin-12 witha vaccine composition comprising at least one antigen-encodingpolynucleotide capable of expression in vivo to produce at least oneBordetella antigen.

In another aspect of the invention, a method is provided for preparingan improved vaccine composition comprising combining a vaccinecomposition comprising at least one Bordetella antigen with aninterleukin-12-encoding polynucleotide capable of expression in vivo toproduce an effective adjuvant amount of interleukin-12.

Another embodiment of the invention provides, in a vaccine compositioncomprising at least one Bordetella antigen and an adjuvant, theimprovement comprising employing as the adjuvant an effective adjuvantamount of interleukin-12. Preferably, the antigen is a Bordetellapertussis antigen, or is lipopolysaccharide, pertussis toxin,filamentous hemagglutinin, or pertactin, or is adsorbed to alum.

The invention also provides, in a vaccine composition comprising anadjuvant and at least one antigen-encoding polynucleotide capable ofexpression in vivo to produce at least one Bordetella antigen, theimprovement comprising employing as the adjuvant an effective adjuvantamount of interleukin-12.

There is also provided as a further embodiment, in a vaccine compositioncomprising at least one Bordetella antigen and an adjuvant, theimprovement comprising employing as the adjuvant aninterleukin-12-encoding polynucleotide capable of expression in vivo toproduce an effective adjuvant amount of interleukin-12.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description of preferredembodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing IL-12 and IFN-γ production by macrophagesstimulated with Bordetella antigens as described in Example 2.

FIG. 2 is a bar graph showing IL-5 and IFN-γ production by spleen cellsfrom mice immunized with Bordetella antigens, then stimulated in vitrowith Bordetella antigens in combination with IL-12, as described inExample 4.

FIG. 3 is a graph showing counts of viable B. pertussis cells in thelungs of mice immunized with B. pertussis whole-cell or acellularvaccines with or without IL-12, then challenged with live B. pertussis,as described in Example 5.

FIG. 4 is a bar graph showing the production of IL-2, IFN-γ, and IL-5 byspleen cells from mice immunized with B. pertussis whole-cell oracellular vaccines with or without IL-12, then stimulated in vitro withBordetella antigens, as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have for the first time demonstrated that theinclusion of IL-12 in an acellular vaccine against Bordetella generatedcell-mediated immune responses similar to those observed with whole-cellvaccines. A type 2 T helper cell (Th2) response normally inducedfollowing immunization of mice with an acellular Bordetella vaccinepreparation can be switched to a Th1/Th0 response by incorporation ofIL-12 into the vaccine formulation. The use of IL-12 as an adjuvant inan acellular pertussis vaccine significantly increased its protectiveefficacy; the rate of B. pertussis clearance from the lungs followingrespiratory challenge was equal to that observed with a potentwhole-cell vaccine. These findings demonstrate a regulatory influence ofIL-12 on the induction of B. pertussis-specific Th1 cells followinginfection or immunization and provides further evidence for the role ofTh1 cells in protective immunity against B. pertussis. The presentinvention provides novel Bordetella vaccine compositions and methods ofadjuvantation of Bordetella vaccines intended to provide a cell-mediatedimmune response against Bordetella by using IL-12 as an adjuvant.

As used herein, Bordetella includes Bordetella pertussis, Bordetellaparapertussis, Bordetella bronchiseptica, and any other Bordetellastrain or isolate that is sufficiently similar so that an immuneresponse (including cell-mediated immunity and/or the generation ofantibodies) raised against antigens present in one isolate will have aneffect against at least some of the other strains or isolates.

A Bordetella antigen includes use of the whole Bordetella organism, awhole organism expressing Bordetella antigens, an antigenic portion ofthe Bordetella organism, recombinantly produced antigen or portionsthereof or fusion proteins comprising antigens, and functionalequivalents of Bordetella antigens. Antigenic portions of Bordetellaorganisms include lipopolysaccharide (LPS), filamentous hemagglutinin(FHA), pertactin, and pertussis toxin (PT). Bordetella LPS is preferablypurified, for example, by gel filtration chromatography. Pertussis toxinmay be active or untreated native pertussis toxin (aPT). Preferably,pertussis toxin may be inactivated pertussis toxin (iPT), such aspertussis toxin inactivated by heat treatment, or detoxified pertussistoxin (PTd), such as pertussis toxin chemically detoxified by treatmentwith formaldehyde. Bordetella antigens may be adsorbed to alum. Inaddition, antigens of the present invention include polynucleotideswhich encode Bordetella antigens. Examples of such polynucleotides arethose comprising the genes for B. perussis FHA (Renauld-Mongenie, G. etal., 1996, PNAS USA 93: 7944-7949) and pertussis toxin (Steffen, P. etal., 1996, EMBO J. 15: 102-109). Other antigenic portions of Bordetellaorganisms that can be used in the compositions and methods of thepresent invention can be determined by those of ordinary skill in theart.

Interleukin-12 (IL-12), originally called natural killer cellstimulatory factor, is a heterodimeric cytokine described, for example,in M. Kobayashi et al., 1989, J. Exp. Med. 170: 827. IL-12 can bepurified from natural sources, produced by chemical synthesis, orpreferably produced by recombinant DNA techniques, for example by theexpression and isolation of IL-12 protein in recombinant host cells asdescribed in detail in International Patent Application WO90/05147,published May 17, 1990 (also European Patent Application No. 441,900),incorporated by reference herein. The DNA and amino acid sequences ofthe 30 kD and 40 kD subunits of the heterodimeric human IL-12 areprovided in the above recited international application and in U.S. Pat.No. 5,571,515, incorporated by reference herein. Research quantities ofrecombinant human and murine IL-12 are also available from GeneticsInstitute, Inc., Cambridge, Mass.

As used herein, “interleukin-12” and “IL-12” refer to interleukin-12,its individual subunits, fragments thereof which exhibit IL-12 adjuvantactivity, polynucleotides encoding IL-12, and functional equivalents of“interleukin-12” and “IL-12”.

Functional equivalents of Bordetella antigens and IL-12 include modifiedBordetella antigens and IL-12 protein such that the resulting Bordetellaantigen or IL-12 product has the same antigenic or adjuvant activity,respectively, as described herein, and polynucleotide sequences thatthrough the degeneracy of the genetic code encode Bordetella antigens orIL-12 polypeptides having the antigenic or IL-12 adjuvant activity,respectively, as described herein. For example, a functional equivalentof a Bordetella antigen or IL-12 can contain a “silent” codon or aminoacid substitution (for example, substitution of an acidic amino acid foranother acidic amino acid, or substitution of a codon for a hydrophobicamino acid for another codon for a hydrophobic amino acid).

Fragments of Bordetella antigens and IL-12 are also encompassed by thepresent invention. Preferably, such fragments retain the desiredantigenic or adjuvant activity or modify it to create a desiredactivity. Fragments of Bordetella antigens or IL-12 may be in linearform or they may be cyclized using known methods, for example, asdescribed in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992)and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253(1992), both of which are incorporated herein by reference. TheBordetella antigens and IL-12 polypeptides provided herein also includeantigens and IL-12 polypeptides characterized by amino acid sequencessimilar to those of purified antigens and IL-12 polypeptides but intowhich modifications are naturally provided or deliberately engineered.For example, modifications in the antigen or IL-12 polypeptide orantigen- or IL-12-encoding polynucleotide sequences can be made by thoseskilled in the art using known techniques. Modifications of interest inthe Bordetella antigen or IL-12 polypeptide sequences may include thealteration, addition, insertion, deletion, mutation, substitution,replacement, or modification of a selected amino acid residue in thecoding sequence. As one example, an additional amino acid may be addedto the N-terminus of the antigen or of IL-12. Also, the amino acidsequence of the antigen or of IL-12 may be altered using random mutationtechniques. It is also possible to attach to antigens or to IL-12 othermoieties, including without limitation carbohydrates, lipids, orpolyethylene glycol, or to remove or alter such moieties. Techniques forsuch alterations, additions, insertions, deletions, mutations,substitutions, replacements, or modifications are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, addition, insertion, deletion, mutation, substitution,replacement, or modification retains the desired activity of theBordetella antigen or IL-12, or modifies it to create a desiredactivity.

The invention also encompasses allelic variants of disclosed Bordetellaantigen- and IL-12-encoding polynucleotides; that is,naturally-occurring alternative forms of isolated polynucleotides whichalso encode antigens or IL-12 polypeptides which are identical,homologous, or related to that encoded by the isolated polynucleotides.

Administration and Dosing

The IL-12 and the Bordetella antigen can be administered as aprophylactic vaccine to hosts, preferably to mammalian hosts, which areeither infected or uninfected with Bordetella. The IL-12 and the antigencan also be administered as a therapeutic vaccine to infected hosts andcan result in amelioration or elimination of the disease state due toinfection by Bordetella organisms.

The amount of Bordetella antigen used in the compositions and methods ofthe present invention is an amount which produces an effectiveimmunostimulatory response in the host. An effective adjuvant amount ofIL-12 is an amount such that when administered it results in an enhancedimmune response relative to the immune response when IL-12 is notadministered. Such amounts of IL-12 will depend on the nature of theBordetella antigen and the dosage amounts of the antigen. In addition,the amount of Bordetella antigen and IL-12 administered to the host willvary depending on a variety of other factors, including the antigen(s)employed, the size, age, body weight, general health, sex, and diet ofthe host, the time or duration of administration, and the particularqualities of the Bordetella infection being treated or vaccinatedagainst. As one example, an effective adjuvanting amount of IL-12polypeptide is desirably between about 0.1 μg to about 0.5 mg of IL-12polypeptide per about 25 μg of antigen. The effective adjuvant amountfor any particular vaccine or antigen will be readily defined bybalancing the efficacy and toxicity of the IL-12 and antigencombination. Adjustment and manipulation of established dose ranges arewell within the ability of those skilled in the art.

In the method of the present invention, an effective adjuvant amount ofIL-12 is administered in combination with a Bordetella antigen, at atime closely related to immunization with the Bordetella antigen, sothat an enhanced immune response is produced relative to an immunizationin which IL-12 is not administered. Thus, the IL-12 can be administeredprior to and preferably just prior to immunization, at the time ofimmunization (i.e. simultaneously), or after immunization (i.e.subsequently). If the IL-12 is administered before the vaccinecomposition, it is desirable to administer it about one or more daysbefore the vaccine. In addition, the IL-12 can be administered prior toimmunization with the Bordetella antigen, followed by subsequentinjections of IL-12 after immunization with the antigen.

The IL-12 and the Bordetella antigen can be administered to a host in avariety of ways. The routes of administration include intradermal,transdermal (for example, by slow-release polymers), intramuscular,intraperitoneal, intravenous, subcutaneous, oral, aural, epidural, analor vaginal (for example, by suppositories), and intranasal routes. Anyother convenient route of administration can be used, for example,infusion or bolus injection, or absorption through epithelial ormucocutaneous linings. In addition, the IL-12 and the Bordetella antigencan be administered in combination with other components or biologicallyactive agents, such as other known adjuvants, (for example, alum, MPL,QS21), pharmaceutically acceptable surfactants such as glycerides,excipients such as lactose, carriers diluents, and vehicles. If desired,certain sweetening, flavoring, and/or coloring agents can also be added.

When used as an adjuvant for a vaccine composition containing aBordetella antigen, IL-12 is desirably admixed as part of the vaccinecomposition itself, and administered by the same route as the vaccinalBordetella antigen. Alternatively, the adjuvanting effect of IL-12 maybe employed by administering IL-12 separately from the vaccinecomposition. When separately administered, the IL-12 is desirably in thepresence of a suitable carrier, such as saline and optionallyconventional pharmaceutical agents enabling gradual release of IL-12.The amount of IL-12 used in this mode of vaccination is similar to theranges identified above when IL-12 is part of the vaccine composition.

Further, Bordetella antigens and/or IL-12 can be administered by in vivoexpression in the host of polynucleotides encoding at least oneBordetella antigen and/or IL-12. Polynucleotides encoding IL-12 or afragment thereof may be used as an adjuvant. The polynucleotides,preferably in the form of DNA, may be delivered to the vaccinated hostfor in vivo expression of Bordetella antigens and/or IL-12. So-called‘naked DNA’ may be used to express Bordetella antigens and/or IL-12 invivo in a host. (Cohen, J., 1993, Science 259: 1691-1692; Fynan, E. etal., 1993, PNAS USA 90: 11478-11482; and Wolff, J. A. et al., 1991,Biotechniques 11:474-485 describe similar uses of ‘naked DNA’, allincorporated by reference herein.) For example, polynucleotides encodingIL-12 or fragments thereof may be incorporated, or transduced, into theBordetella organism itself, if the whole Bordetella organism is to beemployed as the vaccinal antigen. In another example, polynucleotidesencoding Bordetella antigens may be incorporated or transduced intocells of another organism, such that the Bordetella antigens areexpressed on the surface of the cells that may then be employed as thevaccinal antigen. Alternatively, polynucleotides encoding IL-12 orfragments thereof may be administered as part of the Bordetella vaccinecomposition or separately but contemporaneously with the vaccineantigen, for example, by injection.

Still other modes of delivering Bordetella antigens and/or IL-12 to thehost in the form of polynucleotides encoding them are known to those ofskill in the art and may be employed rather than administration ofBordetella antigens and/or IL-12 polypeptides, as desired. For example,polynucleotides encoding IL-12 may be administered as part of a vectoror as a cassette containing the sequences encoding the Bordetellaantigens and/or IL-12 operatively linked to a promoter sequence. (Forexample, see International Patent Application PCT WO94/01139, publishedJan. 20, 1994 and incorporated by reference herein.) Briefly, the DNAencoding the Bordetella antigens and/or IL-12 protein or desiredfragments thereof may be inserted into a nucleic acid cassette. Thiscassette may be engineered to contain, in addition to the antigen orIL-12 sequence to be expressed, other optional flanking sequences whichenable its insertion into a vector. This cassette may then be insertedinto an appropriate vector downstream of a promoter, an mRNA leadersequence, an initiation site, and other regulatory sequences capable ofdirecting the replication and expression of that sequence in vivo.Additional regulatory sequences may be inserted downstream of the codingsequence to be expressed. This vector permits in vivo expression of theBordetella antigens and/or IL-12 polypeptides within the host. WhenIL-12 polynucleotides are employed as the adjuvant, these polynucleotidesequences may be operatively linked to polynucleotide sequences whichencode the Bordetella antigen(s).

IL-12 may be preferable to known adjuvants because of its enhancement ofvaccine efficacy when cell-mediated immunity is required. IL-12 has anadvantage over alum as a Bordetella vaccine adjuvant, as alum inducesTh2 T helper cells rather than the Th1 cells induced by IL-12. Thus,alum-adjuvanted vaccines may be ineffectual for organisms such asBordetella against which a Th1 response is most effective. Additionally,IL-12 is superior to bacterial adjuvants, such as BCG, which may inducein addition to IL-12 other agents or results which may be unanticipatedor uncontrolled. More desirably, IL-12 as an adjuvant should not inducethe uncontrolled production of other cytokines, as do bacterialadjuvants which induce IL-12 along with many other cytokines. Unlikebacterial adjuvants, IL-12 is human in origin and thus unlikely toproduce any sensitization. Moreover, unlike other adjuvants such asIFN-γ or IL-2, IL-12 is relatively stable in vivo. Thus, it isanticipated that IL-12 will be a highly useful adjuvant for use invaccines against Bordetella.

Patent and literature references cited herein are incorporated byreference as if fully set forth.

The following examples illustrate embodiments of the present invention,but are not intended to limit the scope of the disclosure.

EXAMPLE 1 Analysis of Cytokine Production

Mice. Female BALB/c mice were bred and maintained under the guidelinesof the Irish Department of Health. All mice were 8 to 12 weeks old atthe initiation of this and the following experiments.

Cytokine Production. T cell cytokine production was assessed usingspleen cells from mice stimulated with B. pertussis antigens in vitro.Spleen cells (2×10⁶/ml) from immunized or naive control mice werecultured with antigens or with medium alone (background control), andsupernatants were removed after 24 hours to determine IL-2 productionand after 72 hours to determine the concentrations of IFN-γ, IL4, andIL-5. IL-2 release was assessed by the ability of culture supernatantsto support the proliferation of the IL-2-dependent CTLL-2 cell line. Theconcentrations of murine IL-4, IL-5, and IFN-γ were determined byspecific immunoassays using commercially available antibodies(PharMingen, San Diego, Calif., USA) as previously described (B. P.Mahon, K. Katrak, A. Nomoto, A. J. Macadam, P. D. Minor, and K. H. G.Mills, 1995, J. Exp. Med. 181: 1285-1292) and incorporated herein byreference.

The concentration of IL-12 was determined by immunoassays and bioassays.In the immunoassays, commercially available anti-IL-12 monoclonalantibodies C17.8 (rat IgG2a) and C15.6 (rat IgG1) (Genzyme Diagnostics,Cambridge, Mass., USA), which recognize the p40 subunit of murine IL-12as a monomer, a homodimer, or as part of the p70 heterodimer, were usedfor capture and detection respectively. An alkalinephosphotase-conjugated mouse anti-rat IgG1 (PharMingen, San Diego,Calif., USA) was used to detect the second anti-IL-12 antibody. In thebioassays, biologically active IL-12 concentrations were assessed by theability of test supernatants to stimulate the production of IFN-γ bynaive spleen cell preparations. To ensure that the production of IFN-γwas due to the presence of IL-12, test samples were also assayed in thepresence and absence of a specific anti-IL-12 neutralizing antibody (2.5μg/ml of protein G-purified sheep anti-murine IL-12, Genetics Institute,Cambridge, Mass., USA) which can completely neutralize up to 5 ng/ml ofIL-12. Cytokine concentrations were determined by comparing either theproliferation or the OD₄₉₂ for test samples with a standard curve forrecombinant cytokines of known concentration.

EXAMPLE 2 Macrophage Secrete IL-12 in Response to Bordetella Antigens

This experiment tested the ability of killed whole B. pertussis and B.pertussis components to stimulate the production of IL-12 by murinemacrophages.

Macrophages. Murine peritoneal macrophages were obtained from naiveanimals by plastic adherence of cells obtained by peritoneal lavage.Splenic macrophages were prepared by plastic adherence and alveolarmacrophages were isolated by bronchoalveolar lavage as previouslydescribed (K. Redhead, A. Barnard, J. Watkins, and K. H. G. Mills, 1993,Infect. Immun. 61: 3190-3198). The murine macrophage cell line J774 wasalso used in studies of IL-12 production. Macrophages were infected withviable phase I B. pertussis at a bacteria to macrophage ratio of 5:1 fortwo hours before extensive washing. Extracellular bacteria were killedby treatment with polymyxin B sulphate (100 μg/ml) for 40 minutesfollowed by further washing. This treatment reduces the number ofextracelluar bacteria by 5.0 log CFU. Infected macrophages ormacrophages stimulated with heat-inactivated bacteria or bacterialantigens were cultured at 2×10⁵ cells/ml at 37° C. in a 5% CO₂atmosphere. After 24 or 48 hours cell culture supernatants were removedand the production of IL-12 determined by bioassay or immunoassay, asdescribed above in Example 1.

Antigens. The third British reference preparation for pertussis vaccine(88/522) was used as the whole-cell vaccine. Heat-killed B. pertussisfor use in proliferation assays was prepared by incubation of cells at80° C. for 30 min. PT, FHA, and pertactin, prepared from B. pertussisTohama strain, were kindly provided by Carine Capiau at SmithKlineBeecham, Rixensart, Belgium. Chemically detoxified PT (PTd) forimmunization experiments was prepared by treatment with 0.2 to 0.5%formaldehyde for seven days followed by dialysis against PBS containing0.01% formaldehyde. Inactivated PT (iPT) for use in proliferation assayswas prepared by heating active PT at 80° C. for 30 minutes. (Active PTrefers to untreated native PT throughout.) LPS from B. pertussis W28(89/670) was obtained from The National Institute for BiologicalStandards and Control, Potters Bar, Herts, UK. LPS from E. coli(prepared by phenolic extraction and gel filtration chromatography) waspurchased from Sigma Chemical Co., Poole, Dorset, UK.

FIG. 1 shows macrophage production of IL-12 in response to whole B.pertussis and components. IL-12 was tested by immunoassay (A) or bybioassay (B). Results from the immunoassay, which detects p40 and p70,are mean concentrations in supernatants from triplicate cultures ofsplenic macrophages incubated with heat-killed B. pertussis (1×10⁸/mland 5.0×10⁸/ml), B. pertussis LPS (1 μg/ml), E. coli LPS (1 μg/ml), FHA(1 μg/ml), pertactin (1 μg/ml), active PT (1 μg/ml), detoxified PT (PTd,1 μg/ml), or peritoneal macrophages incubated with increasing doses(10⁵-10⁸ CFU/ml) of heat-killed B. pertussis. The bioassay measured theproduction of IFN-γ produced by naive spleen cells incubated for 24hours with supernantants from splenic macrophages (stimuated byincubation with antigen as described for the immunoassay) in thepresence or absence of a polyclonal neutralizing anti-IL-12 antibody at2.5 μg/ml. Levels of IFN-γ produced in the presence of anti-IL-12antibody are only shown where positive responses were observed in theabsence of the antibody and with one dose (5.0×10⁸/ml) of the killedbacteria. Results are means for triplicate assays, and arerepresentative of four independent experiments. Standard deviations wereless than 20% of the mean values.

Adherent cells from the spleens of naive mice stimulated with heatkilled B. pertussis produced significant levels of IL-12, as detected byan immunoassay specific for p40 and p70 (FIG. 1A). Moderate levels ofIL-12 were also detected in supernatants from macrophages incubated withLPS derived from either B. pertussis or another Gram-negative bacteriumE. coli. Tthese levels were enhanced when IFN-γ was added to thecultures (data not shown). In contrast, little or no IL-12 was producedby macrophages stimulated with FHA, PTd, or pertactin, the components ofthe acellular vaccine (FIG. 1A). Peritoneal macrophages also producedIL-12 in response to stimulation with heat-killed B. pertussis in adose-dependent manner (FIG. 1A).

In order to demonstrate that the IL-12 produced was biologically active,we also tested IL-12 production using a bioassay, which measured thestimulation of IFN-γ by murine spleen cells in the presence or absenceof a neutralizing polyclonal anti-IL-12 antibody. Supernatants fromsplenic macrophages that had been stimulated with killed bacteria orpurified LPS induced naive spleen cells to produce high levels of IFN-γ,which was inhibited by the anti-IL-12 antibody (FIG. 1B). Althoughsupernatants from spleen cells stimulated with active PT did stimulatethe production of IFN-γ, this response could not be ablated by theaddition of the anti-IL-12 antibody. Furthermore, IL-12 was not detectedin supernatants of PT-stimulated macrophages using the immunoassay (FIG.1A). Therefore, it is unlikely that active PT induces IL-12 frommacrophages. Active PT is mitogenic for murine T cells and we have foundthat it promotes IFN-γ produced by purified splenic T cells in thepresence of irradiated accessory cells (Ryan and Mill, unpublishedobservations). Therefore, the IFN-γ detected in the IL-12 bioassay usingsupernatants from macrophages stimulated with active PT is likely toresult from direct stimulation of T cells in the spleen cell populationby active PT carried over in the macrophage supernatants.

Live B. pertussis can be taken up by and survive within macrophages, sothe production of IL-12 by macrophages following infection with B.pertussis was also examined. Table 1 shows the secretion of IL-12 bymurine macrophages in response to infection with B. pertussis.Macrophages were infected with B. pertussis for two hours andextensively washed and treated with polymyxin B to kill extracellularbacteria prior to culture in the presence or absence of a neutralizinganti-IL-12 antibody. IL-12 production was assessed using a bioassaywhich measured the production of IFN-γ by naive spleen cells incubatedfor 24 hours with supernatants from infected of control uninfectedmacrophages. Results are expressed as the mean (±SD) IFN-γconcentrations in the supernatants of triplicate cultures measured byimmunoassay. TABLE 1 IFN-γ (pg/ml) Macrophage Infected No antibody +anti-IL-12 Alveolar −   <50 <50 +   700 (41) 100 (16) Peritoneal −   <50<50 + 30,000 (2,245)  75 (31) J774 −   <50 <50 +   900 (66) <50Although the levels of IL-12 are not as high as that observed followingstimulation of peritoneal macrophages with killed bacteria (as in FIG.1A), this may reflect the lower concentration of live bacteria used inthis experiment. Higher levels of viable B. pertussis were employed inother experiments but resulted in cell death of the macrophagepopulations used in vitro. In separate experiments, supernatants ofalveolar, peritoneal, J774, and splenic macrophages removed 24 and 48hours after infection with B. pertussis were also found to contain IL-12detected by the immunoassay (data not shown). Furthermore, peritonealmacrophages recovered from mice 24 hours after interperitoneal injectionwith live B. pertussis secreted significant levels of IL-12 (569 pg perml of culture supernatant in one experiment) without further stimulationin vitro.

EXAMPLE 3 IL-12 Stimulates Immune Cell Proliferation in Response to B.pertussis Antigens

We tested the ability of IL-12 to modulate immune responses to B.pertussis antigens in vivo by immunization of mice with FHA and PTd inthe presence or absence of alum. Spleen cells from immunized or controlmice were tested for in vitro proliferation against heat-killed B.pertussis (10⁶/ml), heat-inactivated PT (1.0 μg/ml), FHA (1.0 μg/ml),and pertactin (1.0 μg/ml) as previously described (K. H. G. Mills, A.Barnard, J. Watkins, and K. Redhead, 1993, Infect. Immun. 61: 399-410)and incorporated herein by reference. Results were calculated as meancounts per minute (CPM) of [³H]thymidine incorporation for triplicatecultures for groups of four to six mice. Stimulation indices werecalculated by dividing the proliferative response to the antigens by theresponse of control cultures, where cells were stimulated with mediumalone.

Recombinant murine IL-12 was kindly provided by Stanley Wolf, GeneticsInstitute, Inc., Cambridge, Mass., USA. Spleen cells from mice immunizedwith soluble or alum-adsorbed FHA and PTd, with or without IL-12 (0.5μg), were stimulated in vitro with iPT (1.0 μg/ml), FHA (5.0 μg/ml), ormedium alone. Proliferative responses were measured by ³H thymidineincorporation after four days and are expressed as counts per minute(CPM) and stimulation indices (SI). The levels of IFN-γ and IL-5 weretested in supernatants after 72 hours of culture. Results are mean (±SD)responses for triplicate cultures for four mice in each group. —, belowthe level of detection. * and **, P<0.01 and P<0.001, respectively,compared to the corresponding value for mice immunized in the absence ofIL-12, determined by Student's t test. TABLE 2 In vitro ProliferationIFN-γ IL-5 Immunization Stimulation CPM SI (ng/ml) (pg(ml) FHA + PTdMedium 118 ± 40  — — FHA 129 ± 53  1.1 ± 0.4 1.8 ± 0.5 — iPT 98 ± 34 0.8± 0.4 2.2 ± 0.8 — FHA + PTd + Medium 1,347 ± 191   — — IL-12 FHA 8,412 ±1491  6.2* ± 0.9  12.3** ± 1.0   — iPT 8,985 ± 3793  6.7* ± 1.6  11.0**± 0.9   — FHA + PTd + Medium 2,380 ± 168   — — alum FHA 13,264 ± 3,010 5.6 ± 2.8 5.7 ± 0.6 410 ± 70  iPT 6,389 ± 2,123 2.7 ± 0.8 12.1 ± 1.3 430 ± 60  FHA + PTd + Medium 1,546 ± 823   — — alum + IL-12 FHA 17,953 ±4,436  11.6 ± 3.4  7.4 ± 3.0 140* ± 30  iPT 7,706 ± 4,128 5.0 ± 3.0 16.3± 2.0  110* ± 20 

Table 2 shows that co-injection with IL-12 augments cellular immuneresponses to B. pertussis antigens. Two weeks after immunization withFHA and PTd in solution, the in vitro proliferative responses of spleencells against the specific antigens were similar to that observedagainst medium alone (Table 2). In contrast, immunization with FHA andPTd in the presence of IL-12 resulted in enhanced proliferativeresponses to FHA, iPT (Table 2) and killed whole bacteria (data notshown). The addition of IL-12 to the alum-adsorbed antigens alsoaugmented the B. pertussis-specific proliferative responses, althoughthis did not reach a level of statistical significance (Table 2).

Co-injection of soluble antigens and IL-12 enhanced the level of IFN-γsecreted in vitro by antigen stimulated spleen cells; IL-5, a Th2 typecytokine, was not detected from spleen cells from these animals (Table2). In contrast, spleen cells from mice immunized with FHA and PTd inthe presence of alum secreted high levels of IL-5 and moderate levels ofIFN-γ, confirming the known effect of alum to favor the induction of Th2type responses in mice. However, co-injection of IL-12 with FHA and PTdadsorbed to alum resulted in a reduction in IL-5 production, but not asignificant increase in the level of IFN-γ secreted, when compared withspleen cells from animals which had received antigens formulated withalum in the absence of IL-12 (Table 2).

EXAMPLE 4 IL-12 Stimulates IFN-γ Production by Immune Cells from MiceImmunized with Bordetella Antigens

Addition of IL-12 in vitro augments IFN-γ production by spleen cellsfrom mice primed for a Th2 response, as shown in FIG. 2. Mice wereimmunized with FHA and PTd in alum and spleen cells were stimulated invitro with FHA, inactivated PT (iPT; 1.0 μg/ml), or medium alone in thepresence of 0, 0.2, and 2.0 ng/ml of recombinant murine IL-12. Thelevels of IFN-γ and IL-5 were tested in spleen cell supernatants after72 hours. Results are expressed as the mean (±SE) cytokine concentrationfor stimulated spleen cells from four mice per group tested intriplicate.

Immunization of mice with FHA and PTd adsorbed to alum generated apotent Th2 response; ex vivo spleen cells produced high levels of IL-5and low levels of IFN-γ following specific antigen stimulation in vitro(FIG. 2). However, the addition of 0.2 or 2.0 ng per ml of recombinantmurine IL-12 to the spleen cells during antigen stimulation in cultureresulted in significantly increased concentrations of IFN-γ andmarginally reduced levels of IL-5 (FIG. 2), demonstrating that IL-12 canmodulate the pattern of in vitro cytokine secretion by in vivo primed Tcells.

EXAMPLE 5 Adjuvant Effect of IL-12 on Immunization with an AcellularPertussis Vaccine

Since we had previously demonstrated that a highly protective whole-cellvaccine induces a Th1 response, we decided to compare the immuneresponses and protection induced with a whole-cell vaccine with anacellular vaccine administered in the presence or absence of IL-12. Inthese studies of the adjuvant effect of IL-12 on immunization with aBordetella pertussis acellular vaccine, groups of 20 mice received twointraperitoneal (i.p.) immunizations four weeks apart with ⅕ a humandose (0.8 IU) of the whole-cell vaccine (88/522), or with an acellularvaccine comprising 5 μg each of FHA, pretactin, and PTd with or withoutrecombinant murine IL-12 (0.5 μg/mouse). Control mice received PBSmedium alone. Two weeks after the second immunization, mice were eithersacrificed to assess immune responses or challenged with B. pertussis.

Aerosol Infection. Respiratory infection of mice was initiated byaerosol challenge using the method originally described by Sato et al.(1980, Infect. Immun. 29: 261-266), with the following modifications. B.pertussis W28 Phase I was grown under agitation conditions at 36° C. inStainer-Scholte liquid medium. Bacteria from a 48-hour culture wereresuspended at a concentration of approximately 2×10¹⁰ colony formingunits (CFU) per ml in physiological saline containing 1% casein. Thechallenge inoculum was administered as an aerosol over a period of 12minutes using a nebulizer directed into an aerosol chamber containinggroups of 20-24 mice. Four mice from each experimental group weresacrificed at 2 hours and at 2, 5, and 9 days after aerosol challenge toassess the number of viable B. pertussis in the lungs.

Enumeration of Viable Bacteria in the Lungs. Lungs were removedaseptically and homogenized in 1 ml of sterile physiological saline with1% casein on ice. 100 μl of undiluted homogenate or of serially dilutedhomogenate from individual lungs were spotted in triplicate onto each ofthree Bordet-Gengou agar plates and the number of CFU was estimatedafter 5 days of incubation. Results are reported as the mean viable B.pertussis for individual lungs from four mice. The limit of detectionwas approximately log₁₀ 0.5 CFU per lung.

BALB/c mice were immunized at 0 and 4 weeks with a whole cell vaccine(WCV), an acellular vaccine (ACV; 5 μg each of soluble PTd, FHA, andpertactin) with or without 0.5 μg IL-12, or PBS alone (Control). Micewere challenged by aerosol inoculation with B. pertussis two weeks afterthe second immunization. The course of respiratory infection wasfollowed by performing viable counts at intervals after challenge.Results are mean (±SE) CFU counts performed on individual lungs intriplicate for four mice per group at each time point.

IL-12 as an adjuvant enhances the protective efficacy of an acellularpertussis vaccine, as shown in FIG. 3. The levels of bacteria in thecontrol mice were still high nine days after challenge (FIG. 3). Thetime course of respiratory infection in mice immunized with the wholecell vaccine was very short with complete clearance by day 5. Bacterialclearance in mice immunized with the acellular vaccine was slower;complete clearance did not occur until day 9 post challenge. However,the addition of IL-12 to the acellular vaccine formulation significantlyenhanced its protective efficacy. Bacterial clearance was complete byday 5 and the bacterial burden on day 2 was lower than that observed inmice immunized with the whole cell vaccine (FIG. 3).

In order to confirm our earlier suggestions on the protective role ofTh1 cells, and to establish that the superior protective efficacyobserved with the acellular vaccine injected with IL-12 was due toenhanced cell mediated immunity, we also tested the immune response ofthe immunized mice on the day of challenge. IL-12 switches the immuneresponse of spleen cells stimulated with an acellular pertussis vaccinefrom a Th2 response to a Th1/Th0 response, as shown in FIG. 4. Mice wereimmunized as described above for FIG. 3 with a whole cell vaccine (WCV),an acellular vaccine (ACV; 5 μg each of soluble PTd, FHA, and pertactin)with or without 0.5 μg IL-12, or PBS alone (Control). The secretion ofIL-2, IFN-γ, and IL-5 was tested following stimulation of spleen cellsfrom immunized mice with iPT (0.2-1.0 μg/ml), FHA (0.2-5.0 μg/ml), andpertactin (0.2-5.0 μg/ml). Results are expressed as the mean (±SE)cytokine concentration to the optimum concentration of antigen forspleen cells from four mice per group tested in triplicate.

Proliferative responses were detected against whole killed B. pertussis,FHA, inactivated PT, and pertactin in spleen cells from mice immunizedwith the acellular vaccine, but these were significantly enhanced in thepresence of IL-12 and approached the levels observed with the whole-cellvaccine (ref 20 and data not shown). An examination of the cytokineprofiles produced by spleen cells stimulated with specific antigen invitro revealed that spleen cells derived from mice immunized with thewhole-cell vaccine secreted IL-2 and IFN-γ but no detectable IL-5 (FIG.4). In contrast, spleen cells from mice which received the acellularvaccine in the absence of IL-12 secreted low levels of IL-2 and IFN-γand low but detectable levels of IL-5. However, spleen cells from miceimmunized with the acellular vaccine in the presence of IL-12 secretedsignificant levels of IL-2 and IFN-γ. Interestingly, IL-12 appeared tohave differential effects on T cells of different antigen specificity,potentiating IL-2 production by T cells specific for FHA and pertactinand IFN-γ production by PT-specific T cells. Overall, the immuneresponse induced with the acellular vaccine incorporating IL-12 as anadjuvant is best described as a mixed Th1/Th2 or Th0 profile.

These significant new findings are that tissue macrophages, includingthose recovered from the lung, spleen, or peritoneal cavity of naivemice, produce IL-12 following exposure to live or killed B. pertussisand that the addition of IL-12 as an adjuvant to a pertussis acellularvaccine enhances its protective efficacy by promoting type 1 T cellcytokine production. We have demonstrated that immunization of mice witha pertussis acellular vaccine comprising PTd, FHA, and pertactinadsorbed to alum, generated a Th2 response in mice and was associatedwith delayed bacterial clearance following respiratory challenge.

1-43. (canceled)
 44. A vaccine composition against Bordetella comprisingat least one Bordetella antigen and an effective adjuvant amount ofinterleukin-12, wherein the composition is capable of eliciting a Th1immune response.
 45. A vaccine composition comprising at least oneBordetella antigen and an effective adjuvant amount of interleukin-12,wherein the composition is capable of stimulating IFN-γ production in ahost immunized with the composition.
 46. The composition of claim 44 or45, wherein the at least one antigen is a Bordetella pertussis antigen.47. The composition of claim 46, wherein the at least one antigen ischosen from lipopolysaccharide, pertussis toxin, filamentoushemagglutinin, and pertactin.
 48. The composition of claim 46, whereinthe at least one antigen is adsorbed to alum.
 49. The composition ofclaim 44 or 45, wherein the at least one Bordetella antigen is encodedby a polynucleotide capable of expressing the at least one Bordetellaantigen in vivo.
 50. The composition of claim 44 or 45, wherein theinterleukin-12 is encoded by a polynucleotide capable of expressing aneffective adjuvant amount of interleukin-12 in vivo.
 51. A method forpreventing, treating, or ameliorating infection by Bordetella in a hostcomprising administering to the host the composition of claim 44 or 45.52. A method for eliciting an immune response against Bordetellacomprising administering simultaneously a first composition comprisingat least one Bordetella antigen and a second composition comprising aneffective adjuvant amount of interleukin-12.
 53. The method of claim 52,wherein the at least one antigen is a Bordetella pertussis antigen. 54.The method of claim 53, wherein the at least one antigen is chosen fromlipopolysaccharide, pertussis toxin, filamentous hemagglutinin, andpertactin.
 55. The method of claim 53, wherein the at least one antigenis adsorbed to alum.
 56. The method of claim 52, wherein the at leastone antigen is administered as a polynucleotide capable of expressingthe at least one antigen in vivo.
 57. The method of claim 52, whereinthe interleukin-12 is administered as a polynucleotide capable ofexpressing interleukin-12 in vivo.
 58. A method for stimulatingclearance of Bordetella from a host comprising administering acomposition comprising at least one Bordetella antigen and an effectiveadjuvant amount of interleukin-12.
 59. The method of claim 58, whereinthe at least one antigen is a Bordetella pertussis antigen.
 60. Themethod of claim 59, wherein the at least one antigen is chosen fromlipopolysaccharide, pertussis toxin, filamentous hemagglutinin, andpertactin.
 61. The method of claim 58, wherein the at least one antigenis adsorbed to alum.
 62. The method of claim 58, wherein the at leastone antigen is administered as a polynucleotide capable of expressingthe at least one antigen in vivo.
 63. The method of claim 58, whereinthe interleukin-12 is administered as a polynucleotide capable ofexpressing interleukin-12 in vivo.